Coronavirions have a rather simple structure. They consist of a nucleocapsid surrounded by a lipid membrane. The helical nucleocapsid is composed of the RNA genome packaged by one type of protein, the nucleocapsid protein N. The viral envelope generally contains 3 membrane proteins: the spike protein (S), the membrane protein (M) and the envelope protein (E). Some coronaviruses have a fourth protein in their membrane, the hemagglutinin-esterase protein (HE). Like all viruses, coronaviruses encode a wide variety of different gene products and proteins.
Most important among these are the proteins responsible for functions related to viral replication and virion structure. However, besides these elementary functions, viruses generally specify a diverse collection of proteins, the function of which is often still unknown, but which are known or assumed to be in some way beneficial to the virus. These proteins may either be essential-operationally defined as being required for virus replication in cell culture- or dispensable. Coronaviruses constitute a family of large, positive-sense RNA viruses that usually cause respiratory and intestinal infections in many different species. Based on antigenic, genetic and structural protein criteria they have been divided into three distinct groups: Group I, II and III. Actually, in view of the great differences between the groups, their classification into three different genera is presently being discussed by the responsible ICTV Study Group. The features that all these viruses have in common are a characteristic set of essential genes encoding replication and structural functions. Interspersed between and flanking these genes, sequences occur that differ profoundly among the groups and that are, more or less, specific for each group.
Of the elementary genes, the most predominant one occupies about two-thirds of the genome. Located at the 5′ end, this so-called “polymerase gene” encodes two large precursors, the many functional cleavage products of which are collectively held responsible for RNA replication and transcription. The other elementary genes specify the basic structural proteins N, M, E, and S. The nucleocapsid (N) protein packages the viral RNA forming the core of the virion. This RNP structure is surrounded by a lipid envelope in which the membrane (M) protein abundantly occurs constituting a dense matrix. Associated with the M protein are the small envelope (E) protein and the spike (S) protein, the latter forming the viral peplomers that are involved in virus-cell and cell-cell fusion. The genes for these structural proteins invariably occur in the viral genome in the order 5′-S-E-M-N-3′.
In infected cells, the coronavirus nucleocapsids are assembled in the cytoplasm. The nucleocapsids interact with the viral envelope proteins which, after their synthesis in the endoplasmic reticulum, accumulate in the intermediate compartment, a membrane system localized between the endoplasmic reticulum (ER) and the Golgi complex. This membrane system acts as the budding compartment: the interaction of the nucleocapsids with the viral envelope proteins leads to the pinching off of virions that are then released from the cell by exocytosis.
We have recently demonstrated that the assembly of coronaviral particles does not require the involvement of nucleocapsids. Particles devoid of a nucleocapsid are assembled in cells when the viral envelope protein genes are co-expressed. The minimal requirements for the formation of virus-like particles (VLPs) are the M and E protein: the S protein is dispensable but is incorporated if present through its interactions with the M protein. Biochemical and electron microscopical analysis revealed that the VLPs are homogeneous in size and have similar dimensions as authentic corona virions. Clearly, the M and E protein have the capacity to associate in the plane of cellular membranes and induce curvature leading to the budding of specific “vesicles” which are subsequently secreted from the cells. An article describing these results has appeared in EMBO Journal (vol. 15, pp. 2020-2028, 1996).
In yet another article, coronavirus like particles were shown which were not devoid of a nucleocapsid, assembly here did not take place independent of a nucleocapsid (Bos et al., Virology 218, 52-60, 1996). Furthermore, packaging of RNA was not very efficient. Furthermore, neither of these two publications provides sufficient targeting and delivery features which would make the VLPs suitable as therapeutic carrier, for example being equipped with specific targeting information and/or with a genetic or nongenetic message.
However, coronaviruses do have several distinct theoretical advantages for their use as vectors over other viral expression systems (see, also, PCT International Publication WO98/49195): (i) coronaviruses are single-stranded RNA viruses that replicate within the cytoplasm without a DNA intermediary, making unlikely the integration of the virus genome into the host cell chromosome; (ii) these viruses have the largest RNA genome known having, in principle, room for the insertion of large foreign genes; (iii) since coronaviruses in general infect the mucosal surfaces, both respiratory and enteric, they may be used to induce a strong secretory immune response; (iv) the tropism of coronaviruses may be modified by the manipulation of the spike (S) protein allowing the engineering of the tropism and virulence of the vector; and, (v) nonpathogenic coronavirus strains infecting most species of interest are available to develop expression systems.
Two types of expression vectors have been developed based on coronavirus genomes. One requires two components (helper dependent) and the other a single genome that is modified either by targeted recombination or by engineering a cDNA encoding an infectious RNA. Helper dependent expression systems, also called minigenomes have been developed using members of the three groups of coronaviruses. Coronavirus derived minigenomes have a theoretical cloning capacity close to 25 kb, since minigenome RNAs of about 3 kb are efficiently amplified and packaged by the helper virus and the virus genome has about 30 kb. This is, in principle, the largest cloning capacity for a vector based on RNA virus genomes. Reverse genetics has been possible by targeted recombination between a helper virus and either nonreplicative or replicative coronavirus derived RNAs (9). Targeted recombination has been mediated by one or two cross-overs. Changes were introduced within the S gene that modified MHV pathogenicity. The gene encoding green fluorescent protein (GFP) was inserted into MHV between genes S and E by targeted recombination, resulting in the creation of a vector with the largest known RNA viral genome (1d). Mutations have also been created by targeted mutagenesis within the E and the M genes showing the crucial role of these genes in assembly.